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With the growth of monoclonal antibodies and other proteins as major modalities in the pharmaceutical industry, there has been an increase in pharmacology and toxicity testing of biotherapeutics in animals. Animals frequently mount an immune response to human therapeutic proteins. This can result in asymptomatic anti-drug antibody formation, immune complexes that affect drug disposition and/or organ function such as kidney, cytokine release responses, fatal hypersensitivity, or a range of reactions in between. In addition, an increasing number of oncology therapeutics are being developed that enhance or directly stimulate immune responses by a variety of mechanisms, which could increase the risk of autoreactivity and an autoimmune-like syndrome in animals and humans. When evaluating the risk of biotherapeutics prior to entering the clinic, the nonclinical safety data may include any of these responses and it is critical to understand whether they represent a safety liability for humans. The DruSafe Leadership group of the IQ Consortium conducted a survey of industry to understand sponsors' experiences with these immune reactions in nonclinical studies related to both immunogenicity and pharmacologically-mediated immune perturbations. The survey covered what pathways were affected, how the immune responses were presented, how the company and health authorities interpreted the data and whether the immune responses were observed in the clinic. Additionally, the survey gathered information on association of these findings with anti-drug antibodies as well as sponsor's use of immunogenicity predictive tools. The data suggests that the ability of a biotherapeutic to activate the immune system, intended or not, plays a significant role on characteristics of the response and whether theys are translatable.

Despite the potential for the chemokine class as therapeutic targets in immune mediated disease, success has been limited. Many chemokines can bind to multiple receptors and many receptors have multiple ligands, with few exceptions. One of those exceptions is CCL20, which exclusively pairs to CCR6 and is associated with several immunologic conditions, thus providing a promising therapeutic target. Following successful evaluation in a single dose, first time in human clinical study, GSK3050002-a humanized IgG1 monoclonal antibody against human CCL20-was evaluated in a 26-week cynomolgus monkey toxicology study. A high incidence of unexpected vascular and organ inflammation was observed microscopically, leading to the decision to halt clinical development. Here we report a dose-responsive increase in the incidence and severity of inflammation in multiple organs from monkeys receiving 30 and 300 mg/kg/week by either subcutaneous or intravenous injection. Histomorphological changes resembled an immune complex-mediated pathology, which is often due to formation of anti-drug antibodies in monkeys receiving a human protein therapeutic and thus not predictive of clinical outcome. However, the presentation was atypical in that there was a clear dose response with a very high incidence of inflammation with a low incidence of ADA that did not correlate well individually. Additionally, the immunohistologic presentation was atypical in that the severity and distribution of tissue inflammation was greater than the numbers of associated immune complexes (i.e., granular deposits). An extensive ex vivo analysis of large molecular weight protein complexes in monkey serum from this study and in human serum samples demonstrated a time-dependent aggregation of GSK3050002, that was not predicted by in vitro assays. The aggregates also contained complement components. These findings support the hypothesis that immune complexes of drug aggregates, not necessarily including anti-drug antibodies, can fix complement, accumulate over time, and trigger immune complex disease. A situation which may have increased clinical relevance than typical anti-drug antibody-associated immune complex disease in monkeys administered human antibody proteins.

Respiratory tract sensitization can have significant acute and chronic health implications. While induction of respiratory sensitization is widely recognized for some chemicals, validated standard methods or frameworks for identifying and characterizing the hazard are not available. A workshop on assessment of respiratory sensitization was held to discuss the current state of science for identification and characterization of respiratory sensitizer hazard, identify information facilitating development of validated standard methods and frameworks, and consider the regulatory and practical risk management needs. Participants agreed on a predominant Th2 immunological mechanism and several steps in respiratory sensitization. Some overlapping cellular events in respiratory and skin sensitization are well understood, but full mechanism(s) remain unavailable. Progress on non-animal approaches to skin sensitization testing, ranging from in vitro systems, -omics, in silico profiling, and structural profiling were acknowledged. Addressing both induction and elicitation phases remains challenging. Participants identified lack of a unifying dose metric as increasing the difficulty of interpreting dosimetry across exposures. A number of research needs were identified, including an agreed list of respiratory sensitizers and other asthmagens, distinguishing between adverse effects from immune-mediated versus non-immunological mechanisms. A number of themes emerged from the discussion regarding future testing strategies, particularly the need for a tiered framework respiratory sensitizer assessment. These workshop present a basis for moving towards a weight-of-evidence assessment.

Profound immunosuppression (e.g., AIDS, transplant therapy) is epidemiologically associated with an increased cancer risk, and often with oncogenic viruses. It is currently unclear how broadly this association translates to therapeutics that modulate immunity. A workshop co-sponsored by the FDA and HESI examined how perturbing the immune system may contribute to carcinogenesis, and highlighted priorities for improving non-clinical risk assessment of targeted immunomodulatory therapies. Conclusions from the workshop were as follows. 1) While profound altered immunity can promote tumorigenesis, not all components of the immune system are equally important in defense against or promotion of cancer and a similar cancer risk for all immunomodulatory molecules should not be assumed. 2) Rodent carcinogenicity studies have limitations and are generally not reliable predictors of cancer risk associated with immunosuppression. 3) Cancer risk needs to be evaluated based on mechanism-based weight-of-evidence, including data from immune function tests most relevant to tumor immunosurveillance or promotion. 4) Information from nonclinical experiments, clinical epidemiology and immunomodulatory therapeutics show that immunosurveillance involves a complex network of cells and mediators. To support a weight-of-evidence approach, an increased focus on understanding the quantitative relationship between changes in relevant immune function tests and cancer risk is needed.

The market for biotherapeutic monoclonal antibodies (mAbs) is large and is growing rapidly. However, attrition poses a significant challenge for the development of mAbs, and for biopharmaceuticals in general, with large associated costs in resource and animal use. Termination of candidate mAbs may occur due to poor translation from preclinical models to human safety. It is critical that the industry addresses this problem to maintain productivity. Though attrition poses a significant challenge for pharmaceuticals in general, there are specific challenges related to the development of antibody-based products. Due to species specificity, non-human primates (NHP) are frequently the only pharmacologically relevant species for nonclinical safety and toxicology testing for the majority of antibody-based products, and therefore, as more mAbs are developed, increased NHP use is anticipated. The integration of new and emerging in vitro and in silico technologies, e.g., cell- and tissue-based approaches, systems pharmacology and modeling, have the potential to improve the human safety prediction and the therapeutic mAb development process, while reducing and refining animal use simultaneously. In 2014, to engage in open discussion about the challenges and opportunities for the future of mAb development, a workshop was held with over 60 regulators and experts in drug development, mechanistic toxicology and emerging technologies to discuss this issue. The workshop used industry case-studies to discuss the value of the in vivo studies and identify opportunities for in vitro technologies in human safety assessment. From these and continuing discussions it is clear that there are opportunities to improve safety assessment in mAb development using non-animal technologies, potentially reducing future attrition, and there is a shared desire to reduce animal use through minimised study design and reduced numbers of studies.

Ionic signaling pathways, including voltage-dependent potassium (Kv) channels, are instrumental in antigen-mediated responses of peripheral T cells. However, how Kv channels cooperate with other signaling pathways involved in T cell activation and differentiation is unknown. We report that multiple Kv channels are expressed by naive CD4+ lymphocytes, and that the current amplitude and kinetics are modulated by antigen receptor–mediated stimulation and costimulatory signals. Currents expressed in naive CD4+ lymphocytes are consistent with Kv1.1, Kv1.2, Kv1.3, and Kv1.6. Effector CD4+ cells generated by optimal TCR and costimulation exhibit only Kv1.3 current, but at approximately sixfold higher levels than naive cells. CD4+ lymphocytes anergized through partial stimulation exhibit similar Kv1.1, Kv1.2, and/or Kv1.6 currents, but approximately threefold more Kv1.3 current than naive cells. To determine if Kv channels contribute to the distinct functions of naive, effector, and anergized T cells, we tested their role in immunoregulatory cytokine production. Each Kv channel is required for maximal IL-2 production by naive CD4+ lymphocytes, whereas none appears to play a role in IL-2, IL-4, or IFN-γ production by effector cells. Interestingly, Kv channels in anergized lymphocytes actively suppress IL-4 production, and these functions are consistent with a role in regulating the membrane potential and calcium signaling.